This invention relates to a composition suitable for administration to mammalian hosts as a therapeutic formulation. More particularly, this invention relates to a combination therapy for free-radical bodily damage employing a lymphokine or cytotoxin such as tumor necrosis factor (TNF) and a biological modifier consisting of either one or more free radical scavengers that protect against damage caused by free-radical generation, or that selectively increase the susceptibility of a tumor to radical damage by depleting or reducing its radical scavenging capacity, or an inhibitor of one or both of the cyclooxygenase or lipoxygenase pathways of arachidonic acid metabolism.
Lymphokines and cytotoxins, such as interleukin-2, interferon-alpha, interferon-gamma, colony stimulating factor, and tumor necrosis factor, are proteins secreted by T cells and/or macrophages upon activation by antigens or lectins. Interleukin-2 (IL-2), a lymphokine which is produced by normal peripheral blood lymphocytes and induces proliferation of antigen or mitogen stimulated T cells after exposure to plant lectins, antigens, or other stimuli, was first described by Morgan, D. A., et al., Science (1976) 193:1007-1008. Then called T cell growth factor because of its ability to induce proliferation of stimulated T lymphocytes, it is now recognized that in addition to its growth factor properties it modulates a variety of functions of immune system cells in vitro and in vivo and has been renamed interleukin-2 (IL-2). IL-2 is one of several lymphocyte-produced, messenger-regulatory molecules which mediate immunocyte interactions and functions.
Tumor necrosis factor (TNF) was first described by Carswell et al., Proc. Natl. Acad. Sci. USA (1975) 72:3666-3670 as an endotoxin-induced serum factor which causes necrosis of chemically transformed tumor cells when growing in mice. Human TNF is known to be cytotoxic to neoplastic cells, and has been produced in recombinant form. See Pennica et al., Nature (London) (1984) 312:724-729 and Shirai et al., Nature (London) (1985) 313:803-806, Wang et al., Science (1985) 228:149-154.
Interferons (IFN) constitute a group of naturally occurring proteins which are known to exhibit anti-viral, anti-tumor and immunoregulatory behavior. Two types of IFN have been identified based on differences in their observed biological properties and molecular structures: Type I and Type II. Beta-interferon (IFN-.beta.) is a Type I IFN which can be induced in fibroblasts by viral challenge and contains about 165 amino acids. IFN-.alpha. is also a Type I IFN inducible in leukocytes, and IFN-.gamma. is a Type II IFN which is induced in lymphocytes in response to specific mitogenic stimuli and contains 146 amino acids.
Combination chemotherapy using two or more anti-cancer drugs to treat malignant tumors in humans is currently in use in research and in the clinic. The anti-cancer drugs may be antimetabolites, alkylating agents, antibiotics, general poisons, etc. Combinations of drugs are administered in an attempt to obtain a synergistic cytotoxic effect on most cancers, e.g., carcinomas, melanomas, lymphomas and sarcomas, and to reduce or eliminate emergence of drug-resistant cells and to reduce side effects to each drug.
For example, it is known that IL-2 may be used with IFN-.gamma. to treat tumor-bearing hosts with synergistic results (European Patent Publication 149,551 published Jul. 24, 1985 (Genentech) and German Patent Publication 3411184 published Oct. 31, 1985 (Deut Roten Kreuzes)) or with augmentation of natural killer activity (Svedersky et al., J. Immunol. (1984), 133:714-718 and Shalaby et al., J. Interferon Res. (1985), 5:571-581). In addition, U.S. Statutory Invention Reg. No. H22, published Feb. 4, 1986 to Creasey et al., discloses a composition exhibiting a synergistic cytotoxic effect in combination therapy of certain breast cancer and myeloma cell lines using synergistically effective amounts of 5-fluorouracil and human recombinant beta-interferon. Furthermore, enhanced anti-tumor activity has been observed using IFN-.gamma. in combination with TNF and chemotherapeutic agents. Svedersky et al., Internl. J. of Immunopharm. (1985) 7:330.
An understanding of the mechanisms of action of various lymphokines and cytotoxins and the basis of tumor cell sensitivity to such proteins would facilitate the clinical investigation and the design of clinical trials of these therapeutic agents. For example, TNF, which is produced primarily by macrophages, has shown an apparent selectivity for many tumor cells, but not normal cells, in its cytotoxic or cytostatic activities. See, e.g., Carswell et al., supra, Wang et al., supra, Ruff and Gifford in Lymphokines, Volume 2, ed. Pick, E. (Academic Press, Inc., NY, N.Y., 1981), pp. 235-272, Beutler and Cerami, Nature (1986) 320:584-588, and Urban et al., Proc. Natl. Acad. Sci. USA (1986) 83:5233-5237, and the references cited therein. The basis for this selective killing of tumor cells is known not to be due to receptor absence, inasmuch as TNF.sup.r cells, such as human diploid fibroblasts, have sufficient numbers of high affinity receptors, internalize TNF, and degrade it in an apparently similar fashion as TNF.sup.s cells do. Tsujimoto, M. et al., Proc. Natl. Acad. Sci. USA (1985) 82:7626-7630.
Interleukin-1 alone has a protective effect in a model of free radical dependent tissue injury. Neta et al., J. Immunol. (1986) 136:2483-2485. In addition, it has been found that TNF-.alpha. and IFN-.gamma. induce neutrophils from normal and chronic granulomatous-disease patients to release superoxide. Palladino et al., Clin. Res. (1986) 34:502 and Palladino et al., Ped. Res. (1986) 20:302.
The biological activity of both oxygen-free radical species and related polyunsaturated fatty acid lipid peroxidation products has been well established. For example, the generation of reactive radical species has been found to be involved in the cytotoxic effects of ionizing radiation (see, e.g., Petkau, Acta. Physiol. Scand. Suppl. (1980) 492:81-90 and Biaglow et al., Radiat. Res. (1983) 95:437-455), various chemotherapeutic agents (see, e.g., Tomasz, Chem. Biol. Interact. (1976) 13:89-97, Lown and Sim, Biochem. Biophys. Res. Commun. (1977) 77:1150-1157 and Borek and Troll, Proc. Natl. Acad. Sci. USA (1983) 80:1304-1307), and a variety of other biological processes, including aging, and the initiation and promotion stages of experimental carcinogenesis (see, e.g., DiGuiseppi and Fridovich, CRC Crit. Rev. Toxicol. (1984) 12:315-342 and Slater, Biochem. J. (1984) 222:1-15). The generation and release of reactive free radicals in the respiratory burst phenomenon used by various cells of the immune system is a well known mechanism of foreign target destruction. See, e.g., Bus and Gibson in Rev. Biochem. Toxicol., eds. Hodgson et al. (Elsevier, North Holland, 1979), pp. 125-149 and Badwey and Karnovsky, Ann. Rev. Biochem. (1980) 49:695-726.
In aerobes, a variety of radical scavenging mechanisms have evolved at both the cellular and organismal level that confer protection from potentially lethal reactive oxygen species, such as the hydroxy radical, superoxide anion, and hydrogen peroxide. See, e.g., DiGuiseppi and Fridovich, supra, Slater, supra, and Bus and Gibson, supra. Importantly, oxygen radicals can initiate longer-lived chain reactions of lipid peroxidation that can be propagated from cell to cell. These peroxidation products are capable of damaging cellular DNA, RNA, protein, and cellular phospholipids. See, e.g., Slater, supra, Bus, supra, Moody and Hassan, Proc. Natl. Acad. Sci. USA (1982) 79:2855-2859, Lesko et al., Biochemistry (1980) 19:3023-3028, and Cerutti et al. in Genes and Proteins in Oncogenesis (Academic Press, NY, 1983), pp. 55-67. The protective cellular mechanisms against this kind of damage include anti-oxidants and radical scavengers in both the lipid (e.g., .alpha.-tocopherol, .beta.-carotene) and aqueous (e.g., glutathione and ascorbic acid) phases of cells, as well as enzymes such as superoxide dismutase and catalase. See, e.g., Fridovich, Science (1978) 201:875-880 and Meister and Anderson, Ann. Rev. Biochem. (1983) 52:711-760. The high plasma uric acid level found in humans has also been shown to be a major radical protective factor. Ames et al., Proc. Natl. Acad. Sci. USA (1981) 78:6858-6862.
Glutathione (GSH) and related cellular sulfhydryl compounds represent one of the major mechanisms of detoxification of electrophilic metabolites of xenobiotics and oxygen/lipid radical species. Meister and Anderson, supra. Inhibition of free radicals is postulated as the way in which certain radioprotectors, such as the free radical scavengers cysteine and GSH, operate. GSH becomes oxidized to contain a dithio group as well as to protein-mixed disulfides, when cells are exposed to oxygen-generating compounds or other oxidative stresses. See Adams et al., J. Pharmacol. Exp. Ther. (1983) 227:749-754. Therefore, the content of oxidized GSH is one important indicator of either the type of damage to which a cell has been exposed or of its ability to protect itself from oxidative damage. Buthionine sulphoximine has been shown to be an inhibitor of GSH biosynthesis. See Minchinton et al., Int. J. Radiation Oncology Biol. Phys. (1984) 10:1261-1264.
A protein called monocyte cell line cytotoxin (MCCT) was characterized and the inhibitory effects of various protease inhibitors and hydrogen peroxide scavengers on MCCT activity were studied. Armstrong et al., J.N.C.I. (1985) 74:1-9. In addition, it was found that various hydroxyl radical scavengers inhibited production of a lymphotoxin. See Kobayashi et al., J. Biochem. (Tokyo) (1984) 95:1775-1782. Finally, methisoprinol, a purine derivative, has been shown to increase the production of lymphotoxin, which is a lymphokine. Norin and Ballet, Allergol. Immunopathol. (1982) 10:109-114.
Marcus et al., Cancer Research, 47:4208:4212 (1987) discloses use of vitamin C and IL-2.
Arrick et al., J. Clin. Invest., 71:258-267 (1983) discloses that inhibition of glutathione synthesis (e.g., by buthionine sulfoximine (BSO)) enhances lysis of tumor cells by antineoplastic agents.
Romine and Kessel, Biochem. Pharmacol. (UK) (1986) 35:3323-3326 discloses the role of intracellular glutathione as a determinant of responsiveness to antitumor drugs.
Ono et al., Br. J. Cancer (UK) (1986) 54:749-754 discloses the combined effect of BSO and cyclophosphamide on murine tumors and bone marrow.
Hamilton et al., Biochem. Pharmacol. (Jul. 15, 1985) 34:2583-2586 discloses the enhancement of adriamycin, melphalen, and cisplatin cytotoxicity in drug-resistant and drug-sensitive carcinoma cell lines by use of BSO.
Andrews et al., Cancer Res. (Dec. 1985 ) 45:6250-6253 discloses the differential potentiation of alkylating and platinating agent cytotoxicity in human ovarian carcinoma cells by glutathione depletion.
Russo et al., Cancer Res. (June 1986) 46:2845-2848 discloses selective modulation of glutathione levels in human normal versus tumor cells and differential response to chemotherapy drugs.
Tew et al., Cancer Treatment Rep. (June 1986) 70:715-720 discloses the relationship of glutathione depletion to the antimitotic properties of estramustine.
Russo et al., Int. J. Radiat. Oncol. Biol. Phys. (Aug. 1986) 12:1347-1354 discloses the roles of intracellular glutathione in antineoplastic chemotherapy.
Dorr et al., Invest. New Drugs (1986) 4:305-313 discloses the cytotoxic effects of glutathione synthesis inhibition by BSO on human and murine tumor cells.
Green et al. Cancer Res. (Nov. 1984) 44:5427-5431 discloses that incubation of cells in the presence of BSO resulted in markedly increased (synergistic) melphalan cytotoxicity, and Ozols, Semin. Oncol. (Sept. 1985) 12:7-11 discloses that BSO increases the cytotoxicity of melphalen and cisplatin.
Ozols et al., Dev. Oncol. (1986) 47:277-293 discloses the effect of BSO on the efficacy of antitumor drugs.
Crook et al., Cancer Res. (1986) 46:5035-5038 discloses that BSO enhances the cytotoxicity of cyclophosphamide. Hodgkiss et al., Biochem. Pharmacol. (1985) 34:2175-2178 discloses use of BSO to enhance the cytotoxicity of nitroaromatic compounds. Somfai-Relle et al., Biochem. Pharmacol. (1984) 33:485-490 discloses that BSO sensitizes murine tumor cells to L-phenylalanine mustard.